Faculty of Chemistry, Lomonosov Moscow State University, Moscow, Russia.
Vet Res Commun. 2024 Aug;48(4):2805-2811. doi: 10.1007/s11259-024-10424-x. Epub 2024 May 25.
Multiplex analysis as an immunochip-in-a well format for simultaneous detection of post-vaccinal antibodies to three poultry infections (Newcastle disease, infectious bronchitis and bursal disease) in one chicken sera was developed. The immunochip had a microarray format printed on the bottom of a standard microtiter plate well and consisted of 36 microspots (d = 400 μm each) with three lines of viral antigens absorbed in a gradient of five decreasing concentrations. Optimization of assay conditions revealed the necessity of careful choice of the reaction buffer due to the high tendency of chicken IgY to exhibit unspecific binding. The best results were obtained for PBS buffer (pH 6.0) supplied with 0.1% Tween 20. Assay results were visualized by a number of coloured microspots that were correlated with the specific antibody titre in the analysed serum. High (> 8000), medium (3000-8000) or low (1000-3000) antibody titre level for each of three infections could be quickly assessed in one probe visually or with the help of smartphone. ELISA results (antibody titres) and visual gradient immunochip results interpretation (high, medium, low antibody level/titre) for 63 chicken sera with multiple levels of post-vaccinal antibodies against Newcastle disease, infectious bronchitis and bursal disease were in good correlation.
建立了一种微芯片-孔板格式的多重分析方法，用于同时检测鸡血清中三种禽类感染（新城疫、传染性支气管炎和传染性法氏囊病）的疫苗后抗体。免疫芯片的微阵列格式印在标准微孔板孔的底部，由 36 个微点（每个直径 400 μm）组成，三条线的病毒抗原以逐渐降低的五个浓度梯度吸收。优化实验条件表明，由于鸡 IgY 具有高度的非特异性结合倾向，因此需要仔细选择反应缓冲液。在 pH 6.0 的 PBS 缓冲液中添加 0.1%吐温 20 时，获得了最佳结果。通过多个显色微点来可视化检测结果，这些微点与分析血清中的特异性抗体滴度相关。通过肉眼或智能手机可以快速评估三种感染中的每一种的高（>8000）、中（3000-8000）或低（1000-3000）抗体滴度水平。63 份鸡血清的 ELISA 结果（抗体滴度）和视觉梯度免疫芯片结果解释（高、中、低抗体水平/滴度）与针对新城疫、传染性支气管炎和传染性法氏囊病的多种疫苗后抗体水平具有良好的相关性。
